A basic property of the immune system is the immunologic tolerance that provides for self/non-self discrimination, so that the immune system can protect the host from external pathogens without reacting against itself. When the immune system reacts against itself, autoimmune disease results. (Annu. Rev. Med. 48:341-351, 1997)
Oral tolerance has been proven to be of therapeutic benefit in autoimmune diseases, including uveitis, collagen-induced arthritis, adjuvant arthritis, systemic lupus erythematosus, multiple sclerosis, thyroiditis, myasthenia gravis, inflammatory bowel disease and diabetes. (Annals of the New York Academy of Scien. ces 778(1): 217-227, 1996)
An orally administered antigen encounters the gut-associated lymphoid tissue (GALT), which is a well-developed immune network. The GALT consists of villi, lamina propria, intraepithelial lymphocytes and Peyer's patch, wherein Peyer's patch consists of lymphoid nodules interspersed among the villi. The epithelium that lines the gut contains M cells for transporting antigens and microorganisms. Antigens and pathogens in the gut can only penetrate the barrier through M cells, specialized to deliver antigen directly to underlying immune cells. Then, the intraepithelial lymphocytes recognizing the antigens translocate to Peyer's patches to trigger immune responses. In Peyer's patches, B cells and macrophages present the antigens to lymphocytes, and then the antigen-specific lymphocytes circulate to the lamina propria in the mucosal system (such as mucosal surface of respiratory tract) of the body. However, M cells are scattered and few, less than 0.1% of epithelial cells. (Science 277:949952, 1997; Science 277:910-911, 1997)
The intestinal immune system includes innate immunity and adaptive immunity for protecting against diseases mediated via oral administration. The portion of tissue infected by pathogens secretes chemokines for inducing the non-specific innate immune responses of macrophages, monocytes, granulocytes and natural killer cells. Macrophages and granulocytes can directly take pathogens by phagocytosis. Natural killer cells are the immune cells in the body that first respond to viral infections or cells infected by other pathogens. Natural killer cells not only attack the target cells, but also secrete cytokines for regulating immune responses such as interferon-gamma (IFN-γ), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), interleukin-5 (IL-5) and interleukin-10 (IL-10). Macrophages and other antigen presenting cells, such as dendritic cells, present antigens to T cells via MHC class II molecules and co-stimulators on the cell surface, so as to further activate immune responses, i.e. cell-mediated immunity and humoral immunity respectively performed by T cells and B cells. Mucosal immunity is mainly regulated by cytokines secreted from CD4+ T cells. Interleukin-4 (IL-4), IL-5, interleukin-6 (IL-6) and IL-10 secreted from type 2 helper T (TH2) cells involve in stimulating B cells for antibody production. TGF-β secreted from type 3 helper T (TH3) cells induces B cells to produce IgA. (Trends Immunol. 22:244-247, 2004; J. Clin. Invest. 106:935-937, 2000)
Mucosal immune responses are not only induced in the gut, but also in the mucosal system of the whole body. Once there are antigen-specific lymphocytes activated in the inductive site-Peyer's patch in intestine, lymphocytes travel through lymph drain and blood to effector sites, including lamina propria and rectum, genital tract, lung and so on. Then, the antibodies are produced and can be detected at these effector sites. This homing phenomenon is called the common mucosal immune system. Due to the common mucosal immune response, the antibodies induced by oral administration are distributed in the whole body. If the antigen is delivered via oral administration, systematic tolerance or IgA, i.e. oral tolerance, is induced. Oral tolerance includes both systematic unresponsiveness and mucosal responsiveness, wherein the former prevents the whole body from autoimmune diseases, and the later induces active suppression of a specific antigen. Therefore, the mucosal immune system tends to inhibit inflammatory immune responses and further enhance the presentation of IgA in the non-inflammatory local mucosa. (Immunol. Today 18:335-343, 1997)
The surface of the gastrointestinal tract is lined by a simple columnar epithelium to form a barrier against the excessive absorption of bacteria, food antigens and large molecules, and moreover the transportation of small molecules is controlled by the tight junction formed in the barrier. The crypts embedded in the connective tissue include stem cells for regenerating the intestinal epithelial cells. In addition to the intestinal epithelia cells, the intestinal stem cells also can differentiate into Goblet cells and enteroendocrine cells for secreting mucin, and into Paneth cells for secreting antimicrobial peptides. In addition to the Paneth cells staying in the stem cells region, the differentiated cells migrate to the top of the villi. (J. Clin. Invest. 105:14931499, 2000; Science 294:2115-2116, 2001)
It is a reasonable suggestion that the intestinal epithelia cells play a critical role for influencing immune responses to various antigens in the intestinal lumen due to the special location of the epithelial cells. The intestinal epithelial cells can express antigen-presenting molecules as antigen-presenting cells (APCs) so as to regulate T-cell responses in the intestinal mucosa. (Immunol. Today 21:123-128, 2000) Antigen presenting molecules expressed by the intestinal epithelial cells include MHC class I, MHC class II and CD1d. (Gastroenterology 124:1420-1431, 2003) The MHC class I and MHC class II are expressed on the basolateral membrane of epithelial cells, wherein MHC class I is responsible for coupling with CD8+ cells, and MHC class II is responsible for coupling with CD4+ cells. CD1d activates NK T cells via glycolipid. The intestinal epithelial cells play a critical role in the mucosal immune system.
In addition to being the physical barrier, the intestinal epithelial cells secrete antimicrobial peptides against microbes in the gastrointestinal tract and provide signals to other cells. (Immunol. Today 21:123-128, 2000; J. Clin. Invest. 95:55-65, 1995) There are specific antimicrobial peptides, defensins. Defensins are 3-5 kD proteins, which include the α-defensin and β-defensin family, which has 8 peptides. These defensins have organ-specific expression patterns in the epithelial cells of oral mucosa, lung and gastrointestinal tract. (Eur. J. Gastroenterol. Hepatol. 13:771-776, 2001; Nat. Rev. Immunol. 3:710-720, 2003) When the gastrointestinal tract is infected, the intestinal epithelial cells express defensins, the defensins function as chemokines to induce NK cells and dendritic cells to the infected areas, and to perform the so-called innate immunity. In addition to the induction of innate immunity, defensins induce the dendritic cells to express co-stimulator (B7.2) through the toll-like receptor 4, so as to induce the proliferation of T cells. Therefore, defensins involve the link between innate immunity and adaptive immunity.
Other antimicrobial proteins, angiogenins, were considered to involve the angiogenesis of cancer cells; however, in 2003, Hooper disclosed that angiogenins are produced by the Paneth cells in the gastrointestinal tract under normal physiological conditions, and stored in the cellular granules. The angiogenins are secreted into the gastrointestinal tract in response to the lipopolysaccharide stimulation. Comparing the Germ free mice and the mice colonized with intestinal bacteria, it is found that angiogenins are significantly increased due to the presence of the intestinal bacteria. Like defensins, the angiogenins are bactericidal and modulated by local environmental conditions encountered at infected sites. (Nat. Immunol. 4:269-273, 2003)
In addition to secreting antimicrobial peptides against pathogen infection, the intestinal epithelial cells produce many signals for regulating the immune responses in the gastrointestinal tract. In order to protect the host by destroying invading microbes, the intestinal epithelial cells should immediately respond to the invading microbes via the defense mechanism. The defense mechanism is known as the innate immunity system, which uses germline-encoded pattern-recognition receptors for the recognition of the macromolecules of the microbial pathogens, such as lipopolysaccharide present in the cell wall of Gram-negative bacteria. In recent years, the manner in which receptors activate cells has been disclosed. In adult Drosophila, it is shown that Toll induces antifungal and antibacterial peptides upon infection. Medzhitov and Janeways et al. disclose that in mammals, the activation of Tolllike receptor (TLR) results in induction of cytokines and costimulatory molecules required for the activation of the adaptive immune response. (Cell 91:295-298, 1997) The intracellular signaling pathways activated by TLRs share much in common with IL-1R signaling due to their conserved TIR (Toll/IL-1R homology) domains. When the endotoxin is recognized by TLRs, nuclear translocation of NF-kB is triggered to regulate the transcriptions of other genes involved in immune responses. Subsequently, the proinflammatory cytokine genes are activated, antimicrobial peptides are secreted, and the chemokines, such as IL-8, MIP and MCP-1, are secreted. The macrophages and NK cells are induced by these chemokines so as to translocate to the infected site and to destroy the infected cells. (Cell 91:295-298, 1997; Curr. Opin. Immunol. 14:103-110, 2002) Hence, toll-like receptors not only induce the innate immunity system, but also indirectly induce the adaptive immunity system due to the secretion of proinflammatory cytokines triggered by NF-kB activation.
In human cells, there are at least 10 different TLRs, wherein TLR-2, TLR-4 and TLR-9 are more characterized members of the TLR family. TLRs have been shown to mediate the recognition of many types of pathogens, including bacteria and viruses. TLR-4 is the receptor for Gram-negative bacterial LPS, TLR-2 is the receptor for Gram-positive bacterial peptidoglycan, and TLR-9 is the receptor for the unmethylated and phosphorylated cytosine-guanine oligonucleotide, CpG. (Curr. Opin. Immunol. 14:103-110, 2002) TLR3 can recognize double strand RNA virus, (Nature 433:887-892, 2005) and TLR7 can recognize single strand RNA virus. (Proc Natl Acad Sci USA. 101(15):55985603, 2004) When TLR signal pathway is activated, the innate immunity or antiviral response can be induced for host protection.
In normal intestinal epithelial cells, TLR-3 and TLR-5 are constitutively expressed, while TLR2 and TLR4 are barely detectable. (Infection and Immunity 68:7010-7017, 2000) IL-8 is produced by the epithelial cells in response to the stimulation of the bacterial DNA. (FASEB J. 17:1319-1321, 2003) In intestinal epithelial cells, the bacterial CpG oligonucleotides are the ligands for the TLR-9. Furthermore, the intestinal epithelial cells do not induce inflammation in response to all foreign substances, i.e. the intestinal epithelial cells tolerate commensal microflora, while the intestinal epithelial cells provide danger signals to APCs under potential pathogenic conditions or autoimmune diseases, so as to induce the inflammation formed by T cells mediated by APCs. (Cell 118:229-241, 2004)
Under normal steady-state conditions, recognition of commensal bacteria by TLRs plays a crucial role in protection against gut injury. (Cell 118: 229-241, 2004) But in human inflammatory bowel disease (IBD), a Th1-mediated pathological effect is thought to be due to aberrant mucosal immune response to the microflora. (Gastroenterol. Clin. North Am. 31: 41-62, 2002) These findings reveal that TLRs control mucosal homeostasis between host—commensal.
It is well-known that oral administration of a Chinese herbal extract regulates the development of the immunity. It is very possible the Chinese herbal extract regulates the development of the immunity via the intestinal mucosal immunity. Dendrobium species is considered to be the most precious Chinese herb. A Dendrobium species belongs to an orchid family, and its stem is the mainmedicinal part of the plant. It tastes a little sweet and brackish. Some Chinese medical codices disclose that the Dendrobium species as the curative for some illnesses such as mucosal disorders, stomach disorders and ophthalmic disorders. According to our previous research experience, it appears that Dendrobii Herba is the most curative medicinal species.